Cancer stem cells (CSCs, also called tumor initiating cells) are a rare population of cells present in many tumors. CSCs have the capacity to asymmetrically divide giving rise to nonstem cancer cells (nonCSCs) and more CSCs. CSCs are more resistant to therapy than nonCSCs, and a general observation is that therapy increases the number of CSCs. This likely contributes to the treatment resistance seen in patients with advanced disease. Great effort, therefore, is being placed at targeting CSCs in different cancers. Fas is a death receptor that can induce apoptosis in many tissues, most notably in the cells of the immune system. While Fas can kill tumor cells by inducing apoptosis under certain circumstances, we recently found that knocking down Fas or FasL induces a different form of cell death, necrosis, in all cancer cells. We coined this form of cell death DICE (for death induced by CD95/CD95L elimination). Preliminary data indicate that DICE preferentially affects CSCs suggesting that CSCs are addicted to Fas. Based on the observation that treatment refractory cancer is often enriched in CSCs induction of DICE creates an opportunity to target drug resistant cancers by eliminating CSCs. We hypothesize that Fas acts as an essential survival factor for CSCs and that DICE preferentially targets CSCs. We propose to study three specific aims: Specific Aim 1: Study the role of nonapoptotic Fas signaling, canonical Fas-mediated apoptosis, and DICE induction for CSCs and nonCSCs. Hypothesis: Fas can kill nonstem cancer cells (nonCSCs) but it is essential for the survival of CSCs. Specific Aim 2: Explore the mechanism of how Fas regulates CSC fate. Hypothesis: Fas mediates its CSC promoting activities through activation of STAT1. Specific Aim 3: Determine the effect of DICE induction on human CSCs in vivo. Hypothesis: Fas protects CSCs in primary cancers from death. This proposal will test the function of Fas as a protector of CSCs in breast and ovarian cancer. However, our data on multiple other cancer types (both cell lines and mouse models) suggest that this activity is not limited to women's cancer and may have fundamental significance for all solid cancers. This study will determine whether it is possible to deplete cancers of CSCs by inducing DICE and will begin the exploration of the downstream events that mediate this novel CSC-preserving activity of Fas. This proposal brings together recent findings on the role of Fas in cancer cells, the plasticity of CSCs, the function of miRNAs in CSCs, the role of CSCs in therapy resistance, and the concept of targeting CSCs with the ultimate goal to improve therapy.